Several publications and patent documents are referenced in this application by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and patent documents is incorporated by reference herein.
The plastid genome of higher plants is a circular double-stranded DNA molecule, 120-160 kb in size, which may be present in 1900-50000 copies per leaf cell, which carries about 100 chloroplasts (Bendich 1987; Sugiura 1992). There are several reasons why incorporation of transgenes in the plastid genome may be preferred over incorporation of transgenes into the nuclear genome. The advantages of plastid transformation include: natural containment due to lack of pollen transmission in most crops; high-level protein expression; feasibility of expressing multiple genes from operons; and lack of position effect (Maliga 1993; Maliga et al. 1993; Heifetz 2000; Bock 2001; Heifetz and Tuttle 2001; Maliga 2002). Useful traits expressed in chloroplasts are the Bacillus thuringiensis insecticidal protein (McBride et al. 1995; Kota et al. 1999), herbicide resistance (Daniell et al. 1998; Lutz et al. 2001; Ye et al. 2001) and expression of human somatotropin (Staub et al. 2000).
For almost a decade, plastid transformation was feasible only in tobacco (Nicotiana tabacum). Plastid transformation has recently been extended to Arabidopsis thaliana (Sikdar et al. 1998), potato (Solanum tuberosum) (Sidorov et al. 1999), tomato (Ruf et al. 2001) and progress has been made towards transforming plastids in rice (Khan and Maliga 1999). Plastid transformation in these new species has been very inefficient as compared to tobacco. Clearly, a need exists for improved compositions and methods for expressing transgenes in the plastids of a wider range of higher plant species.